The human breast contains a variety of cell types including luminal and basal epithelial cells that form the ductal tree. These two types of epithelial cells are immunocytochemically distinguishable in tissue sections (1) or in enzymatically dissociated single-cell suspensions (2). Antigenic markers that can distinguish these two cell types would include the epithelial membrane antigen (EMA) and keratin 18 which are predominantly expressed in luminal epithelial cells (1,2) and keratin 14 and .alpha.6 integrin which are specifically expressed in basal epithelial cells (1,3). When the expression of these antigenic markers were examined in primary human breast carcinomas, it was found that the carcinoma cells were similar to the luminal epithelial cells in their expression of antigens (1,3,4). This evidence can be interpreted as indicating that breast carcinomas are primarily derived from luminal epithelial cells or their precursor cells with similar phenotypes.
Most normal human breast epithelial cell cultures were derived either from lactational fluids, which contained cells primarily of luminal origin, or were derived from reduction mammoplasty. Cells from reduction mammoplasty, cultured in the commonly used MCDB 170 (Department of Molecular, Cellular and Developmental Biology, University of Colorado) (5) or DFCI-1 (Dana-Farber Cancer Institute) (6) media, exhibit predominantly basal epithelial cell phenotypes (1,4).
Stem cells are undifferentiated cells capable of (a) proliferation, (b) self-maintenance, (c) the production of a large number of differentiated, functional progeny, (d) regenerating the tissue after injury, and (e) a flexibility in the use of these options (37).
There is a strong interest in identifying the human hematopoietic stem cell. Having possession of the stem cell will allow for identification of growth factors associated with its self-regeneration. In addition, there may be as yet undiscovered growth factors associated (1) with the early steps of dedication of the stem cell to a particular lineage; (2) the prevention of such dedication; and (3) the negative control of stem cell proliferation. The availability of stem cells would be extremely useful in bone marrow transplantation, as well as transplantation of other organs or tissues (e.g., liver regeneration and skin grafting). Stem cells are important and ideal targets for gene therapy, where the inserted genes promote the health of the individual into whom the stem cells are transplanted since the stem cells have longer lifespan. In 1992, an Italian research group performed the first human gene transfer involving stem cells instead of lymphocytes in the hope of curing a 5 year old child with the rare genetic disease, adenosine deaminase deficiency. In addition, the ability to isolate the stem cell may serve in the treatment of Fanconi's anemia, Hodgkin's disease, lymphomas and leukemias (e.g., juvenile chronic myelogenous leukemia), as well as other neoplastic conditions (e.g., breast cancer). Thus, there have been world-wide efforts toward isolating the human hematopoietic stem cell in substantially pure or pure form. See U.S. Pat. No. 5,061,620, column 2, lines 3-22.
U.S. Pat. No. 4,411,990 describes a primary assay of human tumor stem cells. Example 1 discloses the treatment of bone marrow cells and their collection using an appropriate medium which contained fetal calf serum that have been inactivated by heat. The cells were suspended in additional horse serum, which contained antibiotics and various amino acids such as glutamine, asparagine, and the like. Column 10 notes that the application of simple in vitro culture technique for studies of human tumor stem cells from primary explants will prove of clinical importance.
U.S. Pat. No. 5,061,620 describes human hematopoietic stem cell, assigned to SyStemix, Inc. of California. Stem cells are separated from dedicated cells and they are maintained by regeneration in a growth medium. The separation is described in Columns 3 and 4 wherein monoclonal antibodies are useful for identifying markers associated with particular cell lineages and/or stages of differentiation. The antibodies may be attached to a solid support to allow for crude separation. Human stem cells are maintained in a medium for culturing the cells which is described in Column 5, Lines 57 and following which medium includes amino acids, vitamins, fetal calf serum and the like. Pluripotent human stem cell regeneration is discussed at Column 6, Lines 52 and following.
U.S. Pat. No. 5,081,030 describes release of cells from affinity matrices. Positive selection of normal marrow stem cells is performed utilizing a monoclonal antibody which selectively recognizes the progenitor cells.
U.S. Pat. No. 5,087,570 teaches a mammalian stem cell composition which is isolated with monoclonal antibodies. The antibodies are attached to a solid support to allow for separation.
U.S. Pat. No. 5,185,438 pertains to nucleic acids encoding stem cell receptor FLK-2. The patent pertains to methods of stimulating the proliferation of primitive mammalian hematopoietic stem cells comprising contacting the stem cells with a ligand that binds to a receptor protein tyrosine kinases expressed in primitive mammalian hematopoietic cells and not expressed in mature hematopoietic cells. A primitive hematopoietic cell is totipotent that is capable of reconstituting all hematopoietic blood cells in vivo. (Column 5, Lines 5-7) Column 8 and following describe the isolation of nucleic acid molecules and proteins that encode mammalian stem cell receptors. A source of stem cells includes fetal liver, spleen, or thymus cells or adult marrow or brain cells. Example 2 in Column 14 indicates a technique for isolating the stem cells from fetal liver. The separated re-suspended tissue is treated with heat-inactivated fetal bovine calf serum to inhibit trypsin activity. The tissue was resuspended in stromal medium which contains fetal calf serum, human serum, glutamine, sodium pyruvate, non-essential amino acids and a mixture of antibiotics. The sequences for various materials identified in the patent are shown. Similarly related is U.S. Pat. No. No. 5,270,458.
U.S. Pat. No. 5,214,133 teaches SCL, a hematopoietic growth and differentiation factor. SCL is a new human gene. The SCL gene is of interest because it is involved with human stem cell leukemia.
U.S. Pat. No. 5,226,914 describes a method for treating connective tissue disorders. Culturing and passaging of marrow stromal cells are described in Column 9. The medium that was utilized included fetal bovine serum. A substantial list of medium components is described in Columns 5 and 6.
U.S. Pat. No. 5,258,367 is of interest in that it relates to utilizing uteroferrin and rose proteins for stimulating proliferation of hematopoietic cells.
U.S. Pat. No. 5,300,422 describes a screening method for controlling agranulocytosis. There the blood is collected and assayed for stem cell activity to N-desmethylclozapine. The stem cells are identified by specific markers which are identified with monoclonol antibodies.
Cancer Research 47:1634-1645, 1987, describes the use of x-ray lethally-irradiated human fibroblast cell mats to isolate and to culture putative human fetal kidney epithelial stem cells which are deficient in gap junctional intercellular communication. The medium used was a modified Eagle's minimum essential medium with increased amount of amino acids and vitamins, and supplemented with sodium pyruvate and fetal bovine serum.
It is an object of the present invention to describe a substantially purified human breast epithelial cell type which has stem cell characteristics.
It is another object of the present invention to describe a technique to obtain human breast epithelial cells, from reduction mammoplasty, that have stem cell characteristics by using a serum-free defined medium (the MSU-1 medium as defined below) to grow two types of normal human breast epithelial cells (HBEC) (i.e., Type I cells with stem cell and luminal epithelial cell characteristics and Type II cells with basal epithelial cell characteristics) and by separating these two types of cells into substantially pure cultures using two contrasting characters: 1) growth promotion and growth inhibition of Type I and Type II HBEC, respectively, by fetal bovine serum, and 2) late and early attachment on plastic culture dishes of Type I and Type II HBEC, respectively, after trypsinization and subculture.
It is yet another object of the present invention to describe a defined culture medium (the MSU-1 medium) that is comprised of a 1:1 (v/v) mixture of a modified Eagle's MEM and a modified MCDB 153, supplemented with recombinant human epidermal growth factor, insulin, hydrocortisone, human transferrin, 3,3',5-D.L.-triiodo thyronine and 17-.beta. estradiol. The medium supports the growth of both Type I and Type II HBEC.
These and other objects will be described hereinafter.